1. Field of the Invention
The present invention relates to a liposome preparation and more particularly to that entrapping or bearing adriamycin as the effective component.
2. Related Arts
Adriamycin has widely been employed as an effective component for curing malignant tumors. Adriamycin has a positive charge at physiological pH and thus binds to cell surfaces as well as mitochondrial and other cellular membranes, particularly to phospholipids which are negatively charged. The drug shows an accumulative cardiac toxicity, and therefore the dosing amount of the drug has been limited. Further, the drug has a relatively high affinity for living tissues, so that the drug rapidly disappears from the blood after intravenous administration. Therefore, there is a little chance of the drug's being taken up by tumor tissue.
On the other hand, liposomes are the lipid vesicles and prepared by suspending a polar lipid film in an aqueous solution. The liposomes have been classified from a morphological view point into (a) small unilamellar vesicles (small liposomes with a single layer), (b) large unilamellar vesicles (large liposomes with a single layer), and (c) multi lamellar vesicles (liposomes with multi-layers). Each of these liposomes has basically the same structure as cell membranes in the living body and thus has been widely employed as a model for studying the physicochemical structure of the living cell membranes. Since 1970, studies have been made towards the application of the liposomes as vehicles or carriers of drugs or enzymes to the living body, based on the assumption that the liposomes would act as microcapsules of drugs or the like having the following advantages:
1. Protection of the encapsulated agent from various metabolic enzymes,
2. Decrease of undesired side effects appearing in other tissues,
3. Suppression of immune reactions against drugs or the like,
4. Sustentation of pharmacological effect due to slow release,
5. Improvement in arrival to target tissues.
6. Increase of amount of drugs or the like taken into cells,
7. Selective uptake into cytoplasm or lysosomes, and
8. Possibility of remote control on arrival of drugs or the like to tissues by, for instance, warming a specific part of the body from outside, thereby causing release of the contents of the liposomes.
In general, the liposomes are prepared from polar lipids such as phosphatidylcholine, which is easily available and has been extensively studied on its physical characteristics, and cholesterol, for adjusting fluidity of the layer or membrane to be formed, and, if necessary, other lipids for giving a specific organ selectivity to the liposomes. According to a conventional method, the liposomes are prepared by dissolving these lipids in organic solvents, treating the resulting solution with a rotary evaporator to evacuate the solvents and thus leave a thin lipid film on the inner surface of the glass vessel, drying the film sufficiently, adding a drug or enzyme solution to swell the film, shaking the vessel to dislodge the film, sonicating the swollen film under an inert gas atmosphere to form a liposome suspension, and removing free drug or enzyme unentrapped in the liposomes from the suspension with use of ultracentrifugation, gel-filtration method, or the like. The drug, enzyme, or the like are encapsulated in the inner space of the liposomes, and may further be embedded in layer(s) of the liposomes or be adhered to the inner and outer surfaces of the liposome membranes, depending on characteristics of the materials in a liposome preparation.
The inventors have carried out many studies to develop an advantageous method to dose anti-tumor agents inclusive of adriamycin; and as one of the studies, they tried to encapsulate adriamycin by various liposomes.
As a result, it has been found that in the case of the liposomes prepared by adding a solution of adriamycin to a lipid film consisting of phosphatidylcholine and cholesterol, the amount of adriamycin encapsulated by the liposomes can not be made so high, since there is limitation of adriamycin solubility. Therefore, they have further studied the utilization of the positive charge in adriamycin, which charge has been considered to be involved in the cardiac toxicity, but introducing various lipids having negative charges into the liposome layer, and found out that the addition of sulfatide of acidic glycolipid results in the liposomes that effectively entrap adriamycin and that the entrapped adriamycin retains its anti-tumor activity as it were. Also, when adriamycin-entrapping liposomes were given to animals, the concentration of the drug in the blood is kept at a higher level than that in the case in which adriamycin was solely dosed, so that an increased probability of the drug's being taken into tumor tissue could be expected and an accumulation of the drug in the heart can be lowered to decrease its toxicity. This was reported by them at the 26th Meeting of the Japanese Society of Neurology, which was held in Matsue City, Shimane, Japan, on May 24, 1985.
The adriamycin-entrapping liposomes reported at the Meeting had been prepared with lipids of phosphatidylcholine, cholesterol, and sulfatide in a molar ratio of 7:2:1, which has been widely employed for encapsulating drugs, enzymes, or the like. The liposomes are convenient for increasing the amount of adriamycin in the liposomes and for decreasing drug accumulation in the heart; but, as a result of further study, it has been found that this molar ratio of lipids is not always the best one. Namely, the reported liposomes cannot be said to be ones that sufficiently satisfy the following requirements of the ideal liposome preparation which provides an effective and suitable utilization of adriamycin:
(a) The amount of adriamycin entrapped in the liposomes is as large as possible; thus, the amount of lipids dosed will be at its minimum.
(b) The accumulation of adriamycin in the heart is low, so as to suppress the manifestation of cardiac toxicity as much as possible,
(c) The concentration of adriamycin is kept at a level high enough to attain a stable and sustaining manifestation of its pharmacological effect, and
(d) Such requirements (a) to (c) are satisfied with a good balance.